中国小麦花叶病毒运动蛋白基因的克隆、表达及其酵母双杂交筛选研究
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摘要
中国小麦花叶病毒(Chinese wheat mosaic virus,CWMV)是本实验室分离
鉴定并且命名的一种由禾谷多黏菌传播的双组分单链正义RNA杆状植物病毒,
它是真菌传杆状病毒属(Furovirus)的一个新成员。位于CWMV RNAl的3’末
端的ORF 3编码一个约37 kDa的运动蛋白。运动蛋白在介导植物病毒细胞到细
胞的运动过程中发挥重要作用,因此,原核表达此蛋白,制备其特异性抗血清,
并应用酵母双杂交系统筛选与其相互作用的宿主蛋白,有助于阐明CWMV在宿
主细胞间运动的分子机制。
本研究根据Diao等(1999)发表的CWMV的RNAl全序列设计特异性表
达引物,应用RT-PCR技术从小麦病叶中扩增得到长约1 kb的DNA片段。将其
克隆至载体pGEM-T,序列分析表明成功获得全长MP基因。开放阅读框架包括
990个核苷酸,编码329个氨基酸,与已报道的CWMV基因组相应核苷酸序列
(EMBL登录号:AJ012005)同源性为99.6%。将MP基因亚克隆至原核表达载
体构建重组质粒pSBET-MP,SDS-PAGE分析表明,转有pSBET-MP的大肠杆菌
BL21(DE3)pLysS高效表37 kDa蛋白,与MP基因阅读框架的理论推算值37.4
kDa相符。并以含表达产物的凝胶为抗原,按常规方法免疫小鼠,免疫三次后第
三天断头收集血清。Western blot分析表明制得的抗血清是针对MP的多抗,并
且具有很强的专一性。ELISA测定其效价约为1:1200。
用Trizol提取小麦总RNA后并将其纯化成mRNA,应用SMART技术合成
第一链cDNA。然后用5’和3’PCR引物进行LD-PCR扩增,合成双链cDNA,
并与pGADT7-Rec一起转化酵母菌AH 109,构建小麦的酵母双杂交cDNA文库。
经检测,所构文库的独立克隆数为2.3x10~6,文库滴度为8.6x10~8cfu/ml,50%的
文库质粒插入片段大于500 bp,可以用于文库筛选。
将MP基因重组到质粒pGBKT7中,构建酵母双杂交载体pGBKT7-MP,电
击转入酵母菌Y187。Western blot分析表明MP基因在酵母体内能正确表达具有
免疫活性的融合蛋白,并排除对宿主细胞的毒害和自激活作用,可作为酵母双杂
交系统中的“诱饵”质粒。
利用酵母菌Y187(MATa型)和AHl09(MATa型)能融合形成二倍体细
胞的原理,将Y187/pGBKT7-MP与小麦cDNA文库菌AHl09接合,通过营养缺
陷培养和x-α-gal活性分析方法筛选与MP相互作用的阳性候选克隆。Lyticase
法抽提阳性候选克隆的质粒,PCR检测文库质粒中插入片段大小。取插入片段
大于500 bp的文库质粒转化大肠杆菌TGl,提取质粒后通过回转实验进一步排
除假阳性。最终对确定的13个阳性克隆的插入片段进行DNA测序,测序结果
提交GenBank分析,发现共筛得五种蛋白可能与MP具有相互作用,分别是:
交替氧化酶(alternative oxidase,AOX)、硫辛酸合酶类似蛋白(liDoic acid
Chinese wheat mosaic virus (CWMV) is a bipartite RNA virus which was isolated and identified by our laboratory. It is a new member of the genus Furovirus. The 3'-proximal ORF of CWMV RNA1 encodes a 37 kDa movement protein (MP) . The movement protein plays a very pivotal role in viral cell-to-cell movement. So it is important to produce an antiserum against the MP. We also seek for the genes encoding MP-associated proteins from the wheat cDNA library using yeast two-hybrid system. This work will redound to illustrate molecular mechanism about viral cell-to-cell movement.In this study, a pair of primers were designed based on the complete sequence of RNA1 of CWMV. About a 1 kb DNA fragment was amplified from the total RNA of infected wheat leaves by RT-PCR and was cloned into pGEM-T vector. The MP gene was confirmed after the 1 kb DNA fragment was sequenced. The complete MP gene was 990 nts long and encoded 229 amino acid residues. Compared with a reported CWMV MP gene sequence (EMBL: AJ012005) , the homology of nucleotide sequences was 99.6%. The MP gene was subcloned into pSBET. SDS-PAGE analysis showed that a 37 kDa protein which was equal to CWMV MP in molecular weight was highly expressed in E.coli BL21 (DE3) pLysS induced with IPTG. The MP was purified by SDS-PAGE and the antiserum against the protein was raised in mouse. Western blot and ELISA analysis showed the antiserum could combine with prokaryotic expressed protein and the titer was 1: 1200.The total RNA was isolated from wheat with Trizol and was purified into mRNA. The first-strand cDNA was synthesized from the mRNA with SMART technology, and the ds cDNA was further synthesized with 5' and 3' PCR primer by long distance PCR. The yeast strain AH109 was transformed with ds cDNA and pGADT7-Rec in order to construct the cDNA library. The library consists of 2.3×10~6 independent clones, and the titer library is 8.6×10~8 cfu/ml. About half of the clones had the inserted fragments were longer than 500 bp.The recombinant plasmid was constructed by ligating the gene of MP into pGBKT7, and then transformed into yeast Y187 to observe MP expression by western blot. As a result, the MP was expressed as a fusion protein and has immunogenic activity. The plasmid pGBKT7-MP has no toxicity to yeast and can't activate transcription of reporter genes by itself. So it can serve as a bait plasmid of yeast two-hybrid system.Y187 (MATα) can mate with AH109 (MATα) to became a AH109/Y187
diploids. For this reason, Y187/pGBKT7-MP was mated with the pretransformed wheat cDNA lirary yeast strain AH 109, and the putative positive clones were obtained by nutritional selection screeing and X-a-gal assay. The yeast plasmid was isolated from the putative positive clones with lyticase method, and then the AD library inserts were amplified by PCR. The AD plasmids were rescued via transformation of E. coli, whose inserted fragments were longer than 500 bp. These putative positive clones were used to further analysis by using a modified two-hybrid system. At last, only 13 positive clones were confirmed. After sequences the positive clones were provided to the GenBank. It showed that they encoded five proteins including alternative oxidase > lipoic acid synthase-like protein -, large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase and two unknown proteins.
鉴定并且命名的一种由禾谷多黏菌传播的双组分单链正义RNA杆状植物病毒,
它是真菌传杆状病毒属(Furovirus)的一个新成员。位于CWMV RNAl的3’末
端的ORF 3编码一个约37 kDa的运动蛋白。运动蛋白在介导植物病毒细胞到细
胞的运动过程中发挥重要作用,因此,原核表达此蛋白,制备其特异性抗血清,
并应用酵母双杂交系统筛选与其相互作用的宿主蛋白,有助于阐明CWMV在宿
主细胞间运动的分子机制。
本研究根据Diao等(1999)发表的CWMV的RNAl全序列设计特异性表
达引物,应用RT-PCR技术从小麦病叶中扩增得到长约1 kb的DNA片段。将其
克隆至载体pGEM-T,序列分析表明成功获得全长MP基因。开放阅读框架包括
990个核苷酸,编码329个氨基酸,与已报道的CWMV基因组相应核苷酸序列
(EMBL登录号:AJ012005)同源性为99.6%。将MP基因亚克隆至原核表达载
体构建重组质粒pSBET-MP,SDS-PAGE分析表明,转有pSBET-MP的大肠杆菌
BL21(DE3)pLysS高效表37 kDa蛋白,与MP基因阅读框架的理论推算值37.4
kDa相符。并以含表达产物的凝胶为抗原,按常规方法免疫小鼠,免疫三次后第
三天断头收集血清。Western blot分析表明制得的抗血清是针对MP的多抗,并
且具有很强的专一性。ELISA测定其效价约为1:1200。
用Trizol提取小麦总RNA后并将其纯化成mRNA,应用SMART技术合成
第一链cDNA。然后用5’和3’PCR引物进行LD-PCR扩增,合成双链cDNA,
并与pGADT7-Rec一起转化酵母菌AH 109,构建小麦的酵母双杂交cDNA文库。
经检测,所构文库的独立克隆数为2.3x10~6,文库滴度为8.6x10~8cfu/ml,50%的
文库质粒插入片段大于500 bp,可以用于文库筛选。
将MP基因重组到质粒pGBKT7中,构建酵母双杂交载体pGBKT7-MP,电
击转入酵母菌Y187。Western blot分析表明MP基因在酵母体内能正确表达具有
免疫活性的融合蛋白,并排除对宿主细胞的毒害和自激活作用,可作为酵母双杂
交系统中的“诱饵”质粒。
利用酵母菌Y187(MATa型)和AHl09(MATa型)能融合形成二倍体细
胞的原理,将Y187/pGBKT7-MP与小麦cDNA文库菌AHl09接合,通过营养缺
陷培养和x-α-gal活性分析方法筛选与MP相互作用的阳性候选克隆。Lyticase
法抽提阳性候选克隆的质粒,PCR检测文库质粒中插入片段大小。取插入片段
大于500 bp的文库质粒转化大肠杆菌TGl,提取质粒后通过回转实验进一步排
除假阳性。最终对确定的13个阳性克隆的插入片段进行DNA测序,测序结果
提交GenBank分析,发现共筛得五种蛋白可能与MP具有相互作用,分别是:
交替氧化酶(alternative oxidase,AOX)、硫辛酸合酶类似蛋白(liDoic acid
Chinese wheat mosaic virus (CWMV) is a bipartite RNA virus which was isolated and identified by our laboratory. It is a new member of the genus Furovirus. The 3'-proximal ORF of CWMV RNA1 encodes a 37 kDa movement protein (MP) . The movement protein plays a very pivotal role in viral cell-to-cell movement. So it is important to produce an antiserum against the MP. We also seek for the genes encoding MP-associated proteins from the wheat cDNA library using yeast two-hybrid system. This work will redound to illustrate molecular mechanism about viral cell-to-cell movement.In this study, a pair of primers were designed based on the complete sequence of RNA1 of CWMV. About a 1 kb DNA fragment was amplified from the total RNA of infected wheat leaves by RT-PCR and was cloned into pGEM-T vector. The MP gene was confirmed after the 1 kb DNA fragment was sequenced. The complete MP gene was 990 nts long and encoded 229 amino acid residues. Compared with a reported CWMV MP gene sequence (EMBL: AJ012005) , the homology of nucleotide sequences was 99.6%. The MP gene was subcloned into pSBET. SDS-PAGE analysis showed that a 37 kDa protein which was equal to CWMV MP in molecular weight was highly expressed in E.coli BL21 (DE3) pLysS induced with IPTG. The MP was purified by SDS-PAGE and the antiserum against the protein was raised in mouse. Western blot and ELISA analysis showed the antiserum could combine with prokaryotic expressed protein and the titer was 1: 1200.The total RNA was isolated from wheat with Trizol and was purified into mRNA. The first-strand cDNA was synthesized from the mRNA with SMART technology, and the ds cDNA was further synthesized with 5' and 3' PCR primer by long distance PCR. The yeast strain AH109 was transformed with ds cDNA and pGADT7-Rec in order to construct the cDNA library. The library consists of 2.3×10~6 independent clones, and the titer library is 8.6×10~8 cfu/ml. About half of the clones had the inserted fragments were longer than 500 bp.The recombinant plasmid was constructed by ligating the gene of MP into pGBKT7, and then transformed into yeast Y187 to observe MP expression by western blot. As a result, the MP was expressed as a fusion protein and has immunogenic activity. The plasmid pGBKT7-MP has no toxicity to yeast and can't activate transcription of reporter genes by itself. So it can serve as a bait plasmid of yeast two-hybrid system.Y187 (MATα) can mate with AH109 (MATα) to became a AH109/Y187
diploids. For this reason, Y187/pGBKT7-MP was mated with the pretransformed wheat cDNA lirary yeast strain AH 109, and the putative positive clones were obtained by nutritional selection screeing and X-a-gal assay. The yeast plasmid was isolated from the putative positive clones with lyticase method, and then the AD library inserts were amplified by PCR. The AD plasmids were rescued via transformation of E. coli, whose inserted fragments were longer than 500 bp. These putative positive clones were used to further analysis by using a modified two-hybrid system. At last, only 13 positive clones were confirmed. After sequences the positive clones were provided to the GenBank. It showed that they encoded five proteins including alternative oxidase > lipoic acid synthase-like protein -, large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase and two unknown proteins.
引文
[1] 林忠平.走向21世纪的植物分子生物[M].北京:科学出版社,2000.131-445
[2] Deom C M, Olivwer M J, Beachy R N. The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement [J]. Science, 1987, 237:389-394
[3] Karin Seron, Anne-lise Haenni. Vascular movement of plant viruses[J]. MPMI, 1996, 9 (6): 435-442
[4] Yzfira T, Rhee Y, Chen M-H, et al. Nucleic acid transport in plantmicrobe interactions: the molecules that walk through the walls[J]. Annu Rev Microbiol, 2000, 54:187-219
[5] Mushegian A R, Koonin E V. Cell-to-cell movement of plant virus, insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport system[J]. Arch Virol, 1993, 133 (3-4): 239-257
[6] 李毅,陈章良.植物病毒胞间运动的分子生物学和细胞学研究进展[J].植物生理学通讯 1995,31(6):463-469
[7] 张振臣,李大为,于嘉林等.植物病毒细胞间运动及运动蛋白基因介导的抗病性研究进展 [J].2000,8(4):403-408
[8] Melcher U. The '30K' superfamily of viral movement proteins [J]. 2000, 81: 257-266
[9] Saito T, Imai Y, Meshi T, et al. Interviral homologies of the 30K protein of tobamoviruses[J]. Virology, 1988, 167:653
[10] 郑文光,王晓武,高必达.植物病毒胞间运动分子生物学研究进展.中国生物工程杂志,2003,23(2):52-58
[11] Robards A W, Lucas W J. Plasmodesmata[J]. Annu Rev Plant Physiol. Plant tool biol, 1990, 41:369-419
[12] Kim K S, Lee K W. Geminivirus-induced microtubules and their suggested role in cell-to-cell movement [J]. Phytopathology, 1992, 82 (6): 664-669
[13] Citovsky V, et al. The P30 movement protein of tobacoo mosaic virus is a single-strand nucleic acid binding protein [J]. Cell, 1990, 60:637-647
[14] Citovsky V, Zambryski P. How do plant virus nucleic acid move through intercelluar connections[J]? Bio Essays, 1991, 13 (2): 273-279
[15] Carvalho C M, Pouwels, J, Van Lent, et al. The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro [J]. Journal of Virology, 2004, 78 (3): 1591-1594
[16] Wolf S, Deom C M, Beachy R N, et al. Movement protein of tobacco mosaic virus modifies plasmodesmata size exclusion limit[J]. Science, 1989, 246: 377 - 379.
[17] Kasteel D T, Wellink J, Goldbach R W, et al. Isolation and characterization of tubular structures of cowpea mosaic virus[J]. J Gen Virol, 1997, 78 ( 12): 3167-3170
[18] Heinlein M, Padgett H S, Gens J S, et al. Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection[J]. Science, 1995, 270: 1983-1985
[19] Mas P, Beachy R N. Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein[J]. Plant Biology, 2000, 97 (22): 12345-12349
[20] Kahn T W, Lapidot M, Heinlein M, et al. Domains of the TMV movement protein involved in subcelluar localization [J]. Plant J, 1998, 15 (1): 15-25
[21] Dorokhov Y L, Makinen K, Frolova O Y, et al. A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein. FEBS Lett, 1999, 461 (3): 223-228
[22] Chen M H, Sheng J, Hind G, et al. Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-tocell movement. EMBO, 2000, 19 (5): 913-920
[23] Bransom K C, Weiland J J, Tsai C H, et al. Coding density of the turnip yellow mosaic virus genome: roles of the overlapping coat protein and read through coading regions [J]. Virology, 1995,206:403-412
[24] 凌建群,周雪平,李德葆.植物病毒长距离转运的分子机理[J].病毒学报,2000.16(3):285-289
[25] Carrington J C, Kasschau K D, Mahajan S K, et al. Cell-to-cell and long-distance transport of viruses in plants[J]. The Plant Cell, 1996, 8: 1669-1681
[26] Hilf M E, Dawson W O. The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement [J]. Virology, 1993, 193: 106-114
[27] Fenczik C A, Padgeet H S, Holt C A, et al. Mutation analysis of the movement protein of Odontoglossum ringspot virus to identify a host-range determinant [J]. Mol Plant-Microbe Interact, 1995, 8:666-673
[28] Takeda A, Kaido M, Okuno T, et al. The C terminus of the movement protein of Brome mosaic virus controls the requirement for coat protein in cell-to-cell movement and plays a role in long-distance movement [J]. J Gen Virol, 2004, 85 (6): 1751-1761
[29] Cooper B, Lapidot M, Heick J A, et al. A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility [J]. Virology, 1995, 206 (1) :307-313.
[30] Beck D L, Van Dolleweerd C J, Laugh T V, et al. Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block[J]. Proc Natl Acad Sci USA, 1994, 91 (22): 10310-10314.
[31] Tacke E, Salamini F, Rohde W. Genetic engineering of potato for broad-spectrum protection against virus infection [J]. Nat Biotechnol, 1996, 14 (11): 1597-1600
[2] Deom C M, Olivwer M J, Beachy R N. The 30-kilodalton gene product of tobacco mosaic virus potentiates virus movement [J]. Science, 1987, 237:389-394
[3] Karin Seron, Anne-lise Haenni. Vascular movement of plant viruses[J]. MPMI, 1996, 9 (6): 435-442
[4] Yzfira T, Rhee Y, Chen M-H, et al. Nucleic acid transport in plantmicrobe interactions: the molecules that walk through the walls[J]. Annu Rev Microbiol, 2000, 54:187-219
[5] Mushegian A R, Koonin E V. Cell-to-cell movement of plant virus, insights from amino acid sequence comparisons of movement proteins and from analogies with cellular transport system[J]. Arch Virol, 1993, 133 (3-4): 239-257
[6] 李毅,陈章良.植物病毒胞间运动的分子生物学和细胞学研究进展[J].植物生理学通讯 1995,31(6):463-469
[7] 张振臣,李大为,于嘉林等.植物病毒细胞间运动及运动蛋白基因介导的抗病性研究进展 [J].2000,8(4):403-408
[8] Melcher U. The '30K' superfamily of viral movement proteins [J]. 2000, 81: 257-266
[9] Saito T, Imai Y, Meshi T, et al. Interviral homologies of the 30K protein of tobamoviruses[J]. Virology, 1988, 167:653
[10] 郑文光,王晓武,高必达.植物病毒胞间运动分子生物学研究进展.中国生物工程杂志,2003,23(2):52-58
[11] Robards A W, Lucas W J. Plasmodesmata[J]. Annu Rev Plant Physiol. Plant tool biol, 1990, 41:369-419
[12] Kim K S, Lee K W. Geminivirus-induced microtubules and their suggested role in cell-to-cell movement [J]. Phytopathology, 1992, 82 (6): 664-669
[13] Citovsky V, et al. The P30 movement protein of tobacoo mosaic virus is a single-strand nucleic acid binding protein [J]. Cell, 1990, 60:637-647
[14] Citovsky V, Zambryski P. How do plant virus nucleic acid move through intercelluar connections[J]? Bio Essays, 1991, 13 (2): 273-279
[15] Carvalho C M, Pouwels, J, Van Lent, et al. The movement protein of cowpea mosaic virus binds GTP and single-stranded nucleic acid in vitro [J]. Journal of Virology, 2004, 78 (3): 1591-1594
[16] Wolf S, Deom C M, Beachy R N, et al. Movement protein of tobacco mosaic virus modifies plasmodesmata size exclusion limit[J]. Science, 1989, 246: 377 - 379.
[17] Kasteel D T, Wellink J, Goldbach R W, et al. Isolation and characterization of tubular structures of cowpea mosaic virus[J]. J Gen Virol, 1997, 78 ( 12): 3167-3170
[18] Heinlein M, Padgett H S, Gens J S, et al. Changing patterns of localization of the tobacco mosaic virus movement protein and replicase to the endoplasmic reticulum and microtubules during infection[J]. Science, 1995, 270: 1983-1985
[19] Mas P, Beachy R N. Role of microtubules in the intracellular distribution of tobacco mosaic virus movement protein[J]. Plant Biology, 2000, 97 (22): 12345-12349
[20] Kahn T W, Lapidot M, Heinlein M, et al. Domains of the TMV movement protein involved in subcelluar localization [J]. Plant J, 1998, 15 (1): 15-25
[21] Dorokhov Y L, Makinen K, Frolova O Y, et al. A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein. FEBS Lett, 1999, 461 (3): 223-228
[22] Chen M H, Sheng J, Hind G, et al. Interaction between the tobacco mosaic virus movement protein and host cell pectin methylesterases is required for viral cell-tocell movement. EMBO, 2000, 19 (5): 913-920
[23] Bransom K C, Weiland J J, Tsai C H, et al. Coding density of the turnip yellow mosaic virus genome: roles of the overlapping coat protein and read through coading regions [J]. Virology, 1995,206:403-412
[24] 凌建群,周雪平,李德葆.植物病毒长距离转运的分子机理[J].病毒学报,2000.16(3):285-289
[25] Carrington J C, Kasschau K D, Mahajan S K, et al. Cell-to-cell and long-distance transport of viruses in plants[J]. The Plant Cell, 1996, 8: 1669-1681
[26] Hilf M E, Dawson W O. The tobamovirus capsid protein functions as a host-specific determinant of long-distance movement [J]. Virology, 1993, 193: 106-114
[27] Fenczik C A, Padgeet H S, Holt C A, et al. Mutation analysis of the movement protein of Odontoglossum ringspot virus to identify a host-range determinant [J]. Mol Plant-Microbe Interact, 1995, 8:666-673
[28] Takeda A, Kaido M, Okuno T, et al. The C terminus of the movement protein of Brome mosaic virus controls the requirement for coat protein in cell-to-cell movement and plays a role in long-distance movement [J]. J Gen Virol, 2004, 85 (6): 1751-1761
[29] Cooper B, Lapidot M, Heick J A, et al. A defective movement protein of TMV in transgenic plants confers resistance to multiple viruses whereas the functional analog increases susceptibility [J]. Virology, 1995, 206 (1) :307-313.
[30] Beck D L, Van Dolleweerd C J, Laugh T V, et al. Disruption of virus movement confers broad-spectrum resistance against systemic infection by plant viruses with a triple gene block[J]. Proc Natl Acad Sci USA, 1994, 91 (22): 10310-10314.
[31] Tacke E, Salamini F, Rohde W. Genetic engineering of potato for broad-spectrum protection against virus infection [J]. Nat Biotechnol, 1996, 14 (11): 1597-1600